Temperate phages are common and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses infecting mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity, and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages reveals at least five distinct prophage-expressed viral defense systems that interfere with infection of lytic and temperate phages that are either closely-related (homotypic defense) or unrelated (heterotypic defense). Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defense systems include a single-subunit restriction system, a heterotypic exclusion system, and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival, and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, that acts as a highly effective counter-defense system. Prophage-mediated viral defense offers an efficient mechanism for bacterial success in host-virus dynamics, and counter-defense promotes phage co-evolution.

Bacteriophages are the predominant biological entity on the planet. The recent explosion of sequence information has made estimates of their diversity possible. We describe the genomic comparison of 337 fully sequenced tailed phages isolated on 18 genera and 31 species of bacteria in the Enterobacteriaceae. These phages were largely unambiguously grouped into 56 diverse clusters (32 lytic and 24 temperate) that have syntenic similarity over >50% of the genomes within each cluster, but substantially less sequence similarity between clusters. Most clusters naturally break into sets of more closely related subclusters, 78% of which are correlated with their host genera. The largest groups of related phages are superclusters united by genome synteny to lambda (81 phages) and T7 (51 phages). This study forms a robust framework for understanding diversity and evolutionary relationships of existing tailed phages, for relating newly discovered phages and for determining host/phage relationships.

Formation of NADP ؉ from NAD ؉ is catalyzed by NAD kinase (NadK; EC 2.7.1.23). Evidence is presented that NadK is the only NAD kinase of Salmonella enterica and is essential for growth. NadK is inhibited allosterically by NADPH and NADH. Without effectors, NadK exists as an equilibrium mixture of dimers and tetramers (K D ‫؍‬ 1.0 ؎ 0.8 mM) but is converted entirely to tetramers in the presence of the inhibitor NADPH. Comparison of NadK kinetic parameters with pool sizes of NADH and NADPH suggests that NadK is substantially inhibited during normal growth and, thus, can increase its activity greatly in response to temporary drops in the pools of inhibitory NADH and NADPH. The primary inhibitor is NADPH during aerobic growth and NADH during anaerobic growth. A model is proposed in which variation of NadK activity is central to the adjustment of pyridine nucleotide pools in response to changes in aeration, oxidative stress, and UV irradiation. It is suggested that each of these environmental factors causes a decrease in the level of reduced pyridine nucleotides, activates NadK, and increases production of NADP(H) at the expense of NAD(H). Activation of NadK may constitute a defensive response that resists loss of protective NADPH.NAD metabolism ͉ pyridine nucleotides ͉ metabolic control ͉ NADP synthesis ͉ sedimentation equilibrium

The three activities of NadR were demonstrated in purified protein and assigned to separate domains by missense mutations. The N-terminal domain represses transcription of genes for NAD synthesis and salvage. The C-terminal domain has nicotinamide ribose kinase (NmR-K; EC 2.7.1.22) activity, which is essential for assimilation of NmR, converting it internally to nicotinamide mononucleotide (NMN). The central domain has a weak adenylyltransferase (NMN-AT; EC 2.7.7.1) activity that converts NMN directly to NAD but is physiologically irrelevant. This central domain mediates regulatory effects of NAD on all NadR activities. In the absence of effectors, pure NadR protein binds operator DNA (the default state) and is released by ATP (expected to be present in vivo). NAD allows NadR to bind DNA in the presence of ATP and causes repression in vivo. A superrepressor mutation alters an ATP-binding residue in the central (NMN-AT) domain. This eliminates NMN-AT activity and places the enzyme in its default (DNA binding) state. The mutant protein shows full NmR kinase activity that is 10-fold more sensitive to NAD inhibition than the wild type. It is proposed that NAD and the superrepressor mutation exert their effects by preventing ATP from binding to the central domain.

PAS kinase is an evolutionarily conserved serine/threonine protein kinase. Mammalian PAS kinase is activated under nutrient replete conditions and is important for controlling metabolic rate and energy homeostasis. In yeast, PAS kinase acts to increase the synthesis of structural carbohydrate at the expense of storage carbohydrates through phosphorylation of the enzyme UDP-glucose pyrophosphorylase. We have identified two pathways that activate yeast PAS kinase; one is responsive to nutrient conditions while the other is responsive to cell integrity stress. These pathways differentially activate the two PAS kinase proteins in Saccharomyces cerevisiae, Psk1 and Psk2, with Psk1 alone responding to activation by nonfermentative carbon sources. We demonstrate that, in addition to transcriptional effects, both of these pathways post-translationally activate PAS kinase via its regulatory N-terminus. As a whole, this system acts to coordinate glucose partitioning with alterations in demand due to changes in environmental and nutrient conditions.

BackgroundPhage genome analysis is a rapidly growing field. Recurrent obstacles include software access and usability, as well as genome sequences that vary in sequence orientation and/or start position. Here we describe modifications to the phage comparative genomics software program, Phamerator, provide public access to the code, and include instructions for creating custom Phamerator databases. We further report genomic analysis techniques to determine phage packaging strategies and identification of the physical ends of phage genomes.ResultsThe original Phamerator code can be successfully modified and custom databases can be generated using the instructions we provide. Results of genome map comparisons within a custom database reveal obstacles in performing the comparisons if a published genome has an incorrect complementarity or an incorrect location of the first base of the genome, which are common issues in GenBank-downloaded sequence files. To address these issues, we review phage packaging strategies and provide results that demonstrate identification of the genome start location and orientation using raw sequencing data and software programs such as PAUSE and Consed to establish the location of the physical ends of the genome. These results include determination of exact direct terminal repeats (DTRs) or cohesive ends, or whether phages may use a headful packaging strategy. Phylogenetic analysis using ClustalO and phamily circles in Phamerator demonstrate that the large terminase gene can be used to identify the phage packaging strategy and thereby aide in identifying the physical ends of the genome.ConclusionsUsing available online code, the Phamerator program can be customized and utilized to generate databases with individually selected genomes. These databases can then provide fruitful information in the comparative analysis of phages. Researchers can identify packaging strategies and physical ends of phage genomes using raw data from high-throughput sequencing in conjunction with phylogenetic analyses of large terminase proteins and the use of custom Phamerator databases. We promote publication of phage genomes in an orientation consistent with the physical structure of the phage chromosome and provide guidance for determining this structure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3018-2) contains supplementary material, which is available to authorized users.

This article reports the results of studying three novel bacteriophages, JL, Shanette, and Basilisk, which infect the pathogen Bacillus cereus and carry genes that may contribute to its pathogenesis. We analyzed host range and superinfection ability, mapped their genomes, and characterized phage structure by mass spectrometry and transmission electron microscopy (TEM). The JL and Shanette genomes were 96% similar and contained 217 open reading frames (ORFs) and 220 ORFs, respectively, while Basilisk has an unrelated genome containing 138 ORFs. Mass spectrometry revealed 23 phage particle proteins for JL and 15 for Basilisk, while only 11 and 4, respectively, were predicted to be present by sequence analysis. Structural protein homology to wellcharacterized phages suggested that JL and Shanette were members of the family Myoviridae, which was confirmed by TEM. The third phage, Basilisk, was similar only to uncharacterized phages and is an unrelated siphovirus. Cryogenic electron microscopy of this novel phage revealed a T‫9؍‬ icosahedral capsid structure with the major capsid protein (MCP) likely having the same fold as bacteriophage HK97 MCP despite the lack of sequence similarity. Several putative virulence factors were encoded by these phage genomes, including TerC and TerD involved in tellurium resistance. Host range analysis of all three phages supports genetic transfer of such factors within the B. cereus group, including B. cereus, B. anthracis, and B. thuringiensis. This study provides a basis for understanding these three phages and other related phages as well as their contributions to the pathogenicity of B. cereus group bacteria. IMPORTANCEThe Bacillus cereus group of bacteria contains several human and plant pathogens, including B. cereus, B. anthracis, and B. thuringiensis. Phages are intimately linked to the evolution of their bacterial hosts and often provide virulence factors, making the study of B. cereus phages important to understanding the evolution of pathogenic strains. Herein we provide the results of detailed study of three novel B. cereus phages, two highly related myoviruses (JL and Shanette) and an unrelated siphovirus (Basilisk). The detailed characterization of host range and superinfection, together with results of genomic, proteomic, and structural analyses, reveal several putative virulence factors as well as the ability of these phages to infect different pathogenic species.

We identified 9371 tailed phage prophages of 20 known types in reported complete genome sequences of 3298 bacteria in the Salmonella genus. These include 4758 P2 type and 744 P22 type prophages. The latter prophage types were found in the genome sequences of 127 and 24 bacterial host genera, increasing the known host ranges of phages in these groups by 114 and 20 genera, respectively. These prophage nucleotide sequences displayed much more diversity than was previously known from the 48 P2 and 24 P22 type authentic phages whose genomes have been sequenced. More detailed analysis of these prophage sequences indicated that major capsid protein (MCP) gene exchange between tailed phage clusters or types is extremely rare and that P22 prophage-encoded tailspikes correspond perfectly with their hosts’ surface polysaccharide structure; thus, MCP and tailspike sequences accurately predict tailed phage type (and thus lifestyle) and host cell surface polysaccharide structure, respectively.